Obesity has reached epidemic proportions and associated health consequences are alarming, but successful treatment remains a significant challenge, because the underlying causes are complex. In addition to the physiological energy and nutrient needs, external, environmental influences can drive appetite and eating through cognitive and hedonic processes, independently from hunger. Substantial progress has been made in our understanding the homeostatic regulation of food intake and body weight and the integration between physiological and central mechanisms within the hypothalamic and brainstem circuitries. Much less is known about the neural mechanisms mediating environmental influences, yet they are important in health and disease. Cues from the environment can become signals for food through associative learning, and based on that acquired ability can control feeding behavior. These cognitive processes enhance survival when they function in concert with homeostatic control and their stimulatory effects may have been adaptive in the past when energy resources were scarce. The developed world is rich in easily accessible palatable foods and stimulatory effects of omnipresent food cues are maladaptive, as they drive overeating and weight gain. Thus, determining the neural mechanisms underlying this cognitive, non-homeostatic motivation to eat is crucial for potential therapeutic interventions. The core components of the forebrain network underlying cue-induced feeding have been identified and include the basolateral area of the amygdala (BLA), the lateral hypothalamus and orexin/hypocretin (ORX) neurons, and the ventromedial prefrontal cortex (vmPFC). Much remains unknown about the temporal functional connectivity within this network and it is critical to determine which specific circuit and neurotransmitter system is the controller of food motivation at test. The proposed studies will utilize cutting-edge chemogenetic methods, DREADDs and Daun02 inactivation, and precise neuroanatomical and neurochemical techniques to establish a novel vmPFC circuitry with the anterior paraventricular nucleus of the thalamus (PVTa) and ORX receptor 1 signaling is the key controller?an on/off switch within the network?for cognitive food motivation (Aim 1 & 2). Another goal is to determine if this integrative function is mediated by the vmPFC neuronal ensemble plasticity, through dynamic communications with the BLA and PVTa (Aim 3). These mechanisms will be interrogated in behavioral preparations for cue- induced consumption (Aim 1) and persistent food seeking (context-mediated renewal of extinguished responding to food cues; Aim 2) in male and female rats. Sex differences in context renewal of food seeking were recently established and experiments here will test whether the vmPFC and PVTa are sites of sex- specific regulation via connections with the ventral hippocampal formation. The findings from these studies will establish key neurotransmitter and circuitry mechanisms mediating cognitive food motivation and potential sites of sex differences and novel targets for treatment of insatiable appetite and overeating.